Klaus Fritze

A reproducible model of middle cerebral artery occlusion in mice: hemodynamic, biochemical, and magnetic resonance imaging.

A reproducible model of thread occlusion of the middle cerebral artery (MCA) was established in C57 Black/6J mice by matching the diameter of the thread to the weight of the animals. For this purpose, threads of different diameter (80 to 260 μm) were inserted into the MCA of animals of different weights (18 to 33 g), and the success of vascular occlusion was evaluated by imaging the ischemic territory on serial brain sections with carbon black. Successful occlusion of the MCA resulted in a linear relationship between body weight and thread diameter (r = 0.46, P < 0.01), allowing precise selection of the appropriate thread size. Laser-Doppler measurements of CBF, neurological scoring, and 2,3,5-triphenyltetrazolium chloride staining confirmed that matching of animal weight and suture diameter produced consistent cerebral infarction. Three hours after MCA occlusion, imaging of ATP, tissue pH, and cerebral protein synthesis allowed differentiation between the central infarct core, in which ATP was depleted, and a peripheral penumbra with reduced protein synthesis and tissue acidosis but preserved ATP content. Perfusion deficits and ischemic tissue alterations could also be detected by perfusion- and diffusion-weighted magnetic resonance imaging, demonstrating the feasibility of dynamic evaluations of infarct evolution. The use of multiparametric imaging techniques in this improved MCA occlusion model opens the way for advanced pathophysiological studies of stroke in gene-manipulated animals.

Effect of global system for mobile communication (GSM) microwave exposure on blood-brain barrier permeability in rat

Abstract We investigated the effects of global system for mobile communication (GSM) microwave exposure on the permeability of the blood-brain barrier using a calibrated microwave exposure system in the 900 MHz band. Rats were restrained in a carousel of circularly arranged plastic tubes and sham-exposed or microwave irradiated for a duration of 4 h at specific brain absorption rates (SAR) ranging from 0.3 to 7.5 W/kg. The extravasation of proteins was assessed either at the end of exposure or 7 days later in three to five coronal brain slices by immunohistochemical staining of serum albumin. As a positive control two rats were subjected to cold injury. In the brains of freely moving control rats (n = 20) only one spot of extravasated serum albumin could be detected in one animal. In the sham-exposed control group (n = 20) three animals exhibited a total of 4 extravasations. In animals irradiated for 4 h at SAR of 0.3, 1.5 and 7.5 W/kg (n = 20 in each group) five out of the ten animals of each group killed at the end of the exposure showed 7, 6 and 14 extravasations, respectively. In the ten animals of each group killed 7 days after exposure, the total number of extravasations was 2, 0 and 1, respectively. The increase in serum albumin extravasations after microwave exposure reached significance only in the group exposed to the highest SAR of 7.5 W/kg but not at the lower intensities. Histological injury was not observed in any of the examined brains. Compared to other pathological conditions with increased blood-brain barrier permeability such as cold injury, the here observed serum albumin extravasations are very modest and, moreover, reversible. Microwave exposure in the frequency and intensity range of mobile telephony is unlikely to produce pathologically significant changes of the blood-brain barrier permeability.

Effect of global system for mobile communication microwave exposure on the genomic response of the rat brain

The acute effect of global system for mobile communication (GSM) microwave exposure on the genomic response of the central nervous system was studied in rats by measuring changes in the messenger RNAs of hsp70, the transcription factor genes c-fos and c-jun and the glial structural gene GFAP using in situ hybridization histochemistry. Protein products of transcription factors, stress proteins and marker proteins of astroglial and microglial activation were assessed by immunocytochemistry. Cell proliferation was evaluated by bromodeoxyuridine incorporation. A special GSM radiofrequency test set, connected to a commercial cellular phone operating in the discontinuous transmission mode, was used to simulate GSM exposure. The study was conducted at time averaged and brain averaged specific absorption rates of 0.3 W/kg (GSM exposure), 1.5 W/kg (GSM exposure) and 7.5 W/kg (continuous wave exposure), respectively. Immediately after exposure, in situ hybridization revealed slight induction of hsp70 messenger RNA in the cerebellum and hippocampus after 7.5 W/kg exposure, but not at lower intensities. A slightly increased expression of c-fos messenger RNA was observed in the cerebellum, neocortex and piriform cortex of all groups subjected to immobilization, but no differences were found amongst different exposure conditions. C-jun and GFAP messenger RNAs did not increase in any of the experimental groups. 24 h after exposure, immunocytochemical analysis of FOS and JUN proteins (c-FOS, FOS B, c-JUN JUN B, JUN D), of HSP70 or of KROX-20 and -24 did not reveal any alterations. Seven days after exposure, neither increased cell proliferation nor altered expression of astroglial and microglial marker proteins were observed. In conclusion, acute high intensity microwave exposure of immobilized rats may induce some minor stress response but does not result in lasting adaptive or reactive changes of the brain.

Activation tagging: a means of isolating genes implicated as playing a role in plant growth and development

Activation T-DNA tagging has been used to generate a variety of tobacco cell lines selected by their ability to grow either in the absence of auxin or cytokinin in the culture media, or under selective levels of an inhibitor of polyamine biosynthesis. The majority of the cell lines studied in detail contain single T-DNA inserts genetically co-segregating with the selected phenotype. While most of the plants regenerated from the mutant cell lines appear phenotypically normal, several display phenotypes which could be inferred to result from disturbances in the content, or the metabolism, of auxins and cytokinins, or polyamines. The tagging vector is designed to allow the isolation of tagged plant genes by plasmid rescue. Confirmation that the genomic sequence responsible for the selected phenotype has indeed isolated is provided by PEG-mediated protoplast DNA uptake of rescued plasmids followed by selection for protoplast growth under the original selective conditions. Several plasmids have been rescued from the mutant lines which confer on transfected protoplasts the ability to grow either in the absence of auxin or cytokinin in the culture media, or under selective levels of an inhibitor of polyamine biosynthesis. This review describes the background to activation tagging and our progress in characterizing the genes that have been tagged in the mutant lines we have generated.

Monitoring the temporal and spatial activation pattern of astrocytes in focal cerebral ischemia using in situ hybridization to GFAP mRNA: comparison withsgp-2 andhsp70 mRNA and the effect of glutamate receptor antagonists

We investigated the temporo-spatial expression of astrocyte glial fibrillary acidic protein (gfap) and sulfated glycoprotein 2 (sgp-2) mRNAs in comparison to 70-kDa heat shock protein (hsp70) mRNA by in situ hybridisation in rats subjected to permanent occlusion of the middle cerebral artery (MCA). Gfap mRNA started to increase in the cingulate cortex of the lesioned hemisphere 6 h after MCA occlusion and gradually spread over the lateral part of the ipsilateral cortex and the striatum from 12 h to 3 days, peaking at 3 days after MCA occlusion. Gfap mRNA also increased in the contralateral cingulate cortex and corpus callosum at 12 and 24 h. Hsp70 mRNA increased markedly in the ipsilateral cortex adjacent to the ischemic lesion, and slightly within the lesion area from 3 to 24 h and disappeared after 3 days. By 7 days, gfap and sgp-2 mRNAs were increased markedly in the peri-infarct area, and in the ipsilateral thalamus parallel with the delayed neuronal damage, whereas the widespread increase of gfap mRNA in the ipsilateral hemisphere declined. Post-occlusion treatment with the glutamate receptor antagonists MK-801 and NBQX slightly attenuate the induction of gfap but did not qualitatively affect the topical expression pattern. Within the cingulate cortex MK-801 treatment resulted in a significant decrease of the signal intensity at all survival times, reflecting most likely an attenuation of lesion-induced spreading depression like depolarization waves by MK-801. The area of hsp70 expression was reduced by both MK-801 and NBQX, most likely reflecting the decrease of the lesion area by both treatment regimens. Our study thus revealed an early and widespread increase of gfap mRNA in the non-ischemic area including the contralateral hemisphere starting between 3 and 6 h, and a delayed circumscribed expression in the peri-infarct border zone after 1 week. Comparison with the expression of hsp70 mRNA suggests that the absence of an early gfap mRNA induction in the peri-lesion zone reflects an impairment of astrocytic function which may be of importance for infarct growth during the early evolution of the pathological process.

Blood-Brain Barrier Disturbances After rt-PA Treatment of Thromboembolic Stroke in the Rat

We studied the effects of rt-PA (recombinant tissue type-plasminogen activator) treatment on the blood-brain barrier (BBB) after thromboembolic stroke in rat. New MRI methods of diffusion and perfusion imaging to observe the hemodynamic and biophysical effects of thrombolysis were combined with methods for assessment of BBB disturbances. In untreated animals clot embolism produced a rapid drop in MRI perfusion values and the ADC (apparent diffusion coefficient), with subsequent infarction. BBB disturbances, visualised as extravasation of serum proteins on cryostat sections, were manifest in nearly all animals in the borderzone of infarcts. In animals treated with rt-PA 15 min after clot embolism thrombolysis resulted in reperfusion of affected brain regions with subsequent improvement of ADC values. Final lesion size on ADC maps was reduced by 36% relative to untreated animals. However, BBB disturbances were not improved after treatment. To the contrary, rt-PA treated animals showed further regions with serum protein extravasation in the infarcted territories and in distant non-ischemic brain regions. MR imaging with the BBB tracer GdDTPA showed more pronounced and widespread contrast enhancement in the rt-PA treated than in the untreated group. Increased blood-brain barrier disturbances have to be taken into account even when thrombolytic therapy is started very early after the onset of stroke.

Developmental and UV Light Regulation of the Snapdragon Chalcone Synthase Promoter.

Expression directed by the 1.1-kb snapdragon chalcone synthase (CHS) promoter linked to the [beta]-glucuronidase reporter gene has been studied in transgenic tobacco. The pattern of expression of the chimeric gene was compared with the expression of the endogenous CHS genes in tobacco and snapdragon. We demonstrate that expression of the CHS promoter is controlled in both an organ-specific and tissue-specific manner. The highest level of expression was observed in immature seeds. Deletions were used to define regions of the promoter required for expression in roots, stems, leaves, seeds, and flower petals of transgenic plants. We have defined the minimal sequences required for expression in different organs and mapped regions of the promoter that influence expression in either a positive or negative manner. A promoter fragment truncated to -39 activates transcription in roots of 4-week-old seedlings, whereas a fragment extending to -197 bp directs expression in petals and seeds. A positive regulatory element located between -661 and -566 and comprising a 47-bp direct repeat is active in all tissues investigated except petals. UV light-regulated expression in leaves of transgenic tobacco seedlings is dependent on the presence of sequences also required for leaf-specific expression. Within the intact promoter, sequences that individually confer different patterns of expression interact to produce the highly regulated expression pattern of CHS.

Induction of signal transduction pathways through promoter activation.

Publisher Summary This chapter describes the experimental protocols underlying the process of activation tagging. The chapter focuses on the use of this experimental approach to generate mutant cell (and plant) lines able to grow in culture in the absence of auxin, and discusses the general use of this method of tagging to isolate genes whose overexpression results in a selectable phenotype. The creation of dominant mutations through transcriptional activation provides two immediate advantages over other strategies of gene tagging. First, it allows the direct selection for a specific phenotype from among the population of primary transformants. Second, it eases screening for function of rescued plant sequences following transformation of wild-type cells. Coupled with Agrobacterium protoplast cocultivation, activation tagging could also be applied to generate cell lines, and ultimately plant lines, selected for growth under any biochemical selection. This approach can also be used to produce cell lines to grow in the absence of cytokinin or in the presence of selective levels of an inhibitor of polyamine biosynthesis. This suggests that the procedure may have general application. An advantage of the approach is that the selection does not require a detailed understanding of the process under investigation. Hence, the mutations obtained could be applied to study poorly understood processes, such as the molecular basis of plant hormone action for which mutants are not available at the present time.